The measuring of small quantities of fluid has become increasingly important with the development of the microbiological sciences. This stems first from the fact that even small quantities of biological liquids display a high degree of activity. Furthermore, the reseacher and manufacturer frequently have only such miniscular volumes with which to work. In addition, the use of small concentrated samples allows faster reaction rates with the concommitant efficiency in the laboratory.
Consequently, pipetters designed to handle these exceedingly small volumes must do so with a great degree of accuracy. Further, in their design, they should not require a large volume to "prime" them before delivering a small quantity. Moreover, because of the large number of samples occasionally required for different research programs, the design of the pipetter should submit to automation.
Also, the pipetter should not possess pockets or sacks which can trap appreciable quantities of the sample fluid. The secreted fluid, possibly released at subsequent times, can result in deleterious contamination of the samples produced.
Various devices have attempted to satisfy the needs of microbiological measurements. Some of those with greater recency have performed adequately well as parts of particular types of apparatus for which designed.
The capillary tube has represented the classical method for transferring small measured quantities of liquid. Dunking one end of the thin glass tubes allows them to fill through capillary action. Appying to positive gas pressure to the other end will then expel the liquid from the tube into the desired receptacle.
While providing generally acceptable accuracy and precision, the capillary-tube technique suffers from apparent drawbacks. The first, of course, concerns the fact that each measurement requires the handling and control of the device by laboratory personnel; it possesses none of the advantages normally associated with mechanization and automation.
Moreover, while most liquids will rise in the tube under the force of capillary action, the large viscosity of some may prevent them from doing so. Accordingly, the utility of the tubes may not extend to all liquids.
A hand-held and actuated pipetter with disposable fluid-containing tips has provided some assistance in the measuring of small quantities of liquid. Actuating a button with his thumb, the laboratory attendant draws into the tip a predetermined quantity of fuild. Actuating the button a second time releases the fluid from the tip. Again, though, the device requires constant personal attention and does not readily admit of automation.
Sophisticated syringes have also found use in delivering small quantities of fluid. To increase its accuracy, one syringe has had a micrometer screw attached to its piston while providing a digital readout to control is operations. However, purging the system of air represents a significant problem with syringes. The usual technique of pointing a syringe upward to remove the final air bubble provides a serious inconvenience where the device finds use in automated apparatus.
Moreover, the syringe must generally be moved from one or more vials containing the source liquid to a sample tube where it expels the fluid. This movement further limits the utility of the syringe for automated systems. Additionally, dipping the syringe into various solutions allows the deposition of various substances on the outside of its needle. There, it may result in contamination of subsequent fluids with which it makes contact.
In an attempt to ameliorate some of these problems, one company has introduced a syringe in which the piston or plunger extends all the way through both the barrel of the syringe and the needle attached to it. At the base of the needle, where it joins the glass cylinder, the syringe also includes a side opening to the needle shaft. Withdrawing the plunger to its fully retracted position allows the filling of the needle shaft through this side vent. Depressing the plunger various amounts then expels measured quantities of fluid from the tip of the needle.
In order to fill the needle through the side vent, however, requires the plunger to recede to its most retracted position. Consequently, the sample fluid must fill the entire needle. This may require more sample fluid than available at that time. Moreover, where the desired samples do not require the total volume contained in the needle, waste of possibly precious fluid results.
A separate commercial syringe incorporates a hollow plunger within the glass body. This allows the filling of the cylinder through the plunger itself. Again, however, the syringe requires sufficient sample volume to fill the plunger. Not all situations may provide this amount of sample liquid. Moreover, as with the above model, this may result is a substantial waste of precious fluid.
S. T. Nerenberg, in his U.S. Pat. No. 3,184,122, shows a pipette with a two-way glass stopcock and a barrel having a side inlet. Turning the stopcock to a first position fills the tip of the pipette up to the stopcock with a sample fluid. Simultaneously, the barrel of the pipette fills with a second or diluent fluid. With the stopcock in a second position, the sample fluid in the tip exists the pipette into a receptacle followed by a desired amount of the diluent.
To fill the tip of the pipette, however, requires its insertion into the desired fluid with the accompanying possibilities of contamination. Moreover, the tip can not accomodate varying amounts of sample, but only a single preset quantity. Moreover, the apparatus may produce an appreciable waste of the sample fluid during the filling process.
Moreover, the involved liquids contact and, thus, can contaminate the stopcock itself. Also, the amount of liquid entering the areas involved with this internal valving could produce erroneous results for small measured volumes of fluid. Additionally, the tip must move from the sample fluid to the output receptacle during the measuring process. This transition becomes difficult for automated systems to accomodate.
U.S. Pat. No. 3,831,618 to M. D. Liston shows an apparatus processing a capillary probe that forks into two separate capillary conduits. The first line connects to a syringe and contains a silicone oil. The second conduit, filled with a diluent, also connects to a syringe. Withdrawing the silicone oil further into the recesses of the first line allows the ingestion of a sample liquid through the probe. Subsequent shifting of the various liquids will then leave a desired amount of the sample in the common area connecting to both lines. The syringe with the diluent may then expel this fluid into the sample receptacle.
Each of Liston's syringes have pistons under the control of a digital stepping motor. The motor in turn couple through electronic controls to a programable device which directs the behavior of the apparatus.
Liston's device, however, dips its probe into the sample fluid undergoing analysis. This allows the possibility of contaminated or inaccurate samples as discussed with the other systems above. Moreover, Liston does not consider the measuring of different sample liquids while avoiding cross-contamination between them in the first capillary tubing.
W. J. Ambrose et al., in their U.S. Pat. No. 3,612,360, disclose a fluid-handling apparatus with improved valves and piston. The system based on these components automatically transfers quantities of a sample as well as additional liquids into a receptacle. While incorporating significant improvements, the device utilizes a single probe to both ingest and expel fluids. This, of course, will impose the limitations discussed above for pipetters in which the fluids pass both into and out of the system through a common opening. Ambrose et al. also reveal new valves which have worked well with their desired quantities of fluid. Their stated error of not more than one microliter could becomee unacceptable for systems delivering microliter quantities of fluids.
R. E. Thiers incorporates a different type of valving in his U.S. Pat. No. 3,719,087. There, he pinches flexible hosing to control the air and vacuum pressure drawing a fluid into and expelling it from a pipette. However, he has not included it in an automated device using a syringe as the basic measuring component.
Many of the devices described above have advanced and improved the techniques of measuring small quantities of fluid. However, the search continues for apparatus which will accurately perform this function in the microliter range, admit to facile automation, and avoid contamination.